Streptococci

Lab Eight:- Medical Microbiology Prepared by: Luma J. Witwit

Streptococci

Streptococci are Gram positive, non motile, non spore forming catalase negative cocci that occur in pairs or chains, older cultures may lose their Gram-positive character most Streptococci are facultative an aerobes, and some are obligate ( strict ) an aerobes.

Classification Based on Lancefield Proteins:

Rebecca Lancefield, working with various streptococcal species, discovered proteins in the cell wall that were unique to certain organisms. These proteins were labeled Group A, Group B, Group C, and so on through Group M. Currently three Lancefield Groups are of medical importance: Group A, Group B, and Group D.

Of the organisms used in this lab the following correlations apply:
Group A Strep--Streptococcus pyogenes
Group B Strep--Streptococcus agalactiae
Group D Strep--Streptococcus bovis, Enterococcus (Streptococcus) faecalis
Streptococcus pneumoniae does not possess Lancefield proteins and is not classified in one of the Lancefield groups. Viridans streptococci is the term applied to alpha- hemolytic Streptococcus species that lack Lancefield proteins.

Specimens:

Throat swab, pus, sputum, blood, urine, vaginal swab, feaces, peritoneal fluid, gingival swab, carious lesion swab.

Lab diagnosis:

Gram stain
Colonial Morphology on blood agar.
Catalase test.
Blood hemolysis
Optochin disc sensitivity test.
Bile solubility test.
Capsular swelling.
Growth 6.5%NaCl
Bacitracin disc test.
CAMP test

Toxins and enzymes of Strepcocci
• Streptokinase ( fibrinolysin): an active proteolytic enzyme that digests fibrin and other proteins . It is produced by many strains of group A ?-hemolytic streptococci.
• Hemolysin : streptococci produce 3 kinds of hemolysin
• Streptodornase ( streptococcal deoxyribonuclease) depolymerizes DNA.
• Hyaluronidase ( spreading factor ): splits hyaluronic acid, an important component of connective tissue(digest host connective tissue) .
• Streptolysin:
1- Streptolysin (S): is an oxygen – stable cytolysin.
2- Streptolysin (O) is reversibly oxygen – labile cytolysin. Anti-Streptolysin O test (ASO test) or ASO titer.
• Protease: causing soft tissue, necrosis, toxic shock syndrome
Streptococci are classified on the basis of colony Morphology, hemolytic, biochemical reactions and serologic specificity. They are divided into three groups by the type of hemolytic on blood agar:

? - hemolytic ( clear, complete lyses of red cells ): s. Pyogens, s. agalagtiae.
? - hemolytic ( incomplete, green color):s. pumoniae, viridans streptococci.
- hemolyticc ( no hemolytic ). s.faecalis.

Serologic grouping is based on antigenic differences in cell wall carbohydrates ( group A to V ) in cell wall pilli associated protein, and in the polysaccharide capsule.

Bacitracin susceptibility:

Procedures

1. Divide a sheep blood agar plate into four quadrants.
2. Label a quadrant with the name of the organism to be tested.
3. Using a sterile loop aseptically transfer the test organism to the plate and streak the quadrant for confluent growth.
4. Aseptically transfer a bacitracin disc (A disc) to the center of the quadrant. Forceps may be used to position the disc. Gently press the disc to the surface of the agar but do not embed the disc in the agar.
5. Invert the plate and place in the incubator for a minimum of 18 hours.
6. Examine the plate for a zone of inhibition of growth around the disc. When finished discard the plate in the biohazard container.

Interpretation: Any zone of inhibition of growth is considered positive (+) for this test. If a red ring can be seen around the disc this is considered a positive test (S. pyogenes).


Optochin Susceptibility:

This test was done as same as the bacitracin test( used optochin disc).
Interpretation: A growth inhibition zone of 15-30 mm is considered a positive (+) test( S. pnumoniae).

Bile Esculin Hydrolysis:

Both enterococci and non enterococcal sp. of group D are able to hydrolyze esculin in the agar slant , the slant is blacken after (24-48 h ).

Characteristics of Enterococcus:

1. Hydrolysis of esculin.
2. Growth at 45Co
3. presence of 40% bile
4. Tolerance to 6.5% NaCl


Oral Streptococci:

The facultative an aerobes Streptococci are the largest group of bacteria isolated from the oral cavity.
Include S.sanguis, S.salivarus, S.oralis, S.mutans found in .
1. tooth surface.
2. epithelial surface.
3. smooth surface dental decay.
The bacterial infections of the tissue surrounding the teeth cause inflammation of the gingival, periodontal ligament, cementum, and alveolar bone.

Oral Streptococci possess enzymes called glucosyl transferase (GTFS) which are able to break down sucrose into glucose and fructose and utilize the glucose moiety in the synthesis of glucan polymers, or glucan.

Some of the glucan synthesized by S.mutans is water insoluble (mutan ) and contributes the organism s ability to colonize on tooth surfaces.
The S. mutans use of sucrose and other carbohydrates as energy source which leads to acid production which can promote enamel demineralization.

Colony Morphology:

On blood agar plates ( horse blood ) Alpha or gamma. Hemolysis, white shiny to smooth translucent.
On Mitis-Salivarius agar (M-S) Agar is differential medium containing dyes, nutrients, 5% sucrose and growth inhibitors for organisms other than Streptococci.

S. mutans:- pin point to medium size, gray to light or medium blue, the colony is soft to the touch.

S. sanguis:- pin point to medium, translucent to dark blue, shiny colonies are very hard and rubbery to the touch of an inoculating needle.

S. salivarius:- The gum-drop colony, large, circular, entire, pulvinate to highly raised, very mucoid, blue. soft to the touch.

S. oralis:- small to medium , circular, dark blue to opaque black, may be rubbery or soft to the touch. Depressed, flat to slightly raised.